This invention provides a method, devices and machines to uniformly and simultaneously mix in tall vessels in such a manner as to increase the aeration of the liquid. The purpose is to provide a robust method, economical devices and very simple machines to uniformly stir and aerate the contents of one tall vessel or thousands of tall vessels, vials, test tubes or deep well microplate wells (6 well, 12 well, 24 well, 48 well, 96 well, 384 well, 864 well, 1536 well, 10,000 well, xe2x80x9cUxe2x80x9d bottom, xe2x80x9cVxe2x80x9d bottom, PCR and other deep well versions of those microplates) at the same time.
Suspending particulates in liquids, breaking up aggregates, aeration of gases in liquids, dissolving solids in liquids, emulsifying two liquids or mixing liquids together has usually been accomplished in large vessels, bottles, flasks and test tubes by magnetic horizontal spin stirring, orbital shakers, vortexing, rocking platforms, bubblers, vibrators or magnetic tumble stirring. In an effort to miniaturize many of the operations that were done in bottles, flasks and test tubes, scientists have shifted to deep well multi well microplates (24 deep well, 48 deep well, 96 deep well, and 384 deep well). Other deep well plates will undoubtedly follow. These deep wells are characterized by a tall vertical height and a very small air surface to volume ratio, thus making aeration difficult. Deep well microplates are now commonly used in a wide variety of scientific applications to act as mini-reaction or culture vessels for liquid assays. However, because of the combination of the tall height of the column of liquid, small air surface area to volume ratio and the significant attraction of liquid surface tension forces to the walls of small diameter wells (especially in the 48, 96, and 384 deep well microplates) it is not possible to adequately resuspend particulates, aerate gases into liquids, stimulate the growth of microorganisms, break up aggregates, break open cells, emulsify two liquids, mix two liquid reagents, or to dissolve a solid in a liquid by simple agitation on an orbital shaker, vortexer, rocker platform, bubbler or by vibration without generating a force so violent that it would throw the liquids out of the miniature vessels.
Magnetic horizontal spin stirring has been adapted to 96 well microplates by VARIOMAG-USA. They put spinning permanent magnet stirrers into the wells of a 96 well microplate and place the 96 well microplate on a magnetic stirring system with an individual modulated (driving) electromagnetic stir point engineered under the center of each well. The placement of the wells exactly over a stir point is critical so the spinning stirrers don""t hit the well walls and lose synchronization with the driving electromagnetic field, causing them to vibrate in the wells. If they hit the well wall the whole stirrer has to be stopped repositioned and slowly started spinning again. Often when the stir point system is rapidly spinning, the stir magnets in individual wells get out of synchronization with the driving electromagnetic field for no apparent reason or due to the vibration of the stirring action moving the plate. Part of this sensitivity phenomena is due to the physics of the interaction of the magnetic fields of the driving electromagnet and the spinning magnetic stirrer which line up parallel to each other, thus resulting in a weaker magnetic coupling than if the poles were directly opposed to each other. This weak magnetic coupling also is demonstrated if there is a differential viscosity between the liquids of different wells of the same microplate, as this will slow the stirrers in those wells and throw those magnetic stirrers out of synchronization. The cost of this magnetic stir point system is $2,400.00 for each 96 well plate and the cost of the individual stirring magnets for each well is $2.45 thus bringing the total cost to stir each plate to $2,635.00. Furthermore a unique stir point configuration (6, 12, 24, 48, 96, 384, 1536, and 10000 wells) is required for each well configuration of the microplate well format used. Because of these technical and cost considerations this system has not been widely used.
In a previous patent application Ser. No. 09/170,459, now U.S. Pat. No. 6,176,609, we invented and applied xe2x80x9cMagnetic Tumble Stirringxe2x80x9d as a method to stir the contents of microplates. Magnetic Tumble Stirring consists of stainless steel magnetic stir discs, bars and dowels of different dimensions, shapes and with magnetic field orientations through the long axis so that they will provide a vigorous stirring action when they are tumbled end over end through the long axis in a vertical magnetic field(s). By making the magnetic stirrers nearly the same diameter or length as the diameter of the vessel they are stirring or by putting multiple magnetic stirrers into a single vessel a very significant stirring action is effected when they tumble through the magnetic axis. Magnetic Tumble Stirring in the vertical plane is produced by the magnetic poles of stirrers inside vessels being attracted to an opposite vertically oriented drive magnetic field produced by a permanent magnet or an electromagnet under the vessel. This vertical magnetic attraction causes the stirrers to stand on a polar end and then fall when either the drive magnetic field or the vessels are moved laterally in respect to each other and the stirrer can not maintain the vertical orientation because of the wall of the vessel or friction with the well bottom. Although this method works well with microplates that have short to medium columns of liquid above them, it is not as effective with the taller deep well microplates. Furthermore it is does not effectively aerate the liquid.
Another way to provide adequate mixing and aeration in microplates is by pipetting the contents of each well up and down. This can be done manually or by robotic work stations. There are several robotic work stations that will do this pipet mixing operation, but they will just mix 4, 8 or 12 wells at a time and the pipets must be washed or changed between wells. Recently Robbins Scientific introduced the xe2x80x9cHydraxe2x80x9d work station which will pipet and mix 96 wells at a time but it costs xcx9c$30,000 and is still laborious as the pipets must be washed between wells. Furthermore, it would be economically impossible to do continuous mixing of multiple microplates simultaneously by pipetting. In addition, it would be difficult to place a whole large robotic workstation in an incubator if microorganisms were the objects being mixed while culturing. Thus, the miniaturization of continuous mixing processes on a large number of microplates remains impractical until now.
Thus it is the object of the method, devices and machines to provide a very simple process of uniformly mixing and aerating the contents of thousands of vessels, vials, test tubes and deep well microplates. We have made stainless steel magnetic stir balls, discs, bars, and dowels of different dimensions, shapes so that they will fit into any vessel and provide a vigorous stirring action when they are levitated by a magnetic field, then dropped by gravity or pulled down by the same or another magnetic field. By making the magnetic stirrers nearly the same diameter or length as the diameter of the vessel they are stirring or by putting multiple magnetic stirrers into a single vessel, a very significant stirring action is effected when they levitate up and down through the liquid. When the stirrers are levitated through the liquid meniscus it significantly increases the liquid""s surface area and therefor increases aeration. We have discovered that while stainless steel is commonly thought to be non-magnetic, several stainless steels that have been xe2x80x9chardenedxe2x80x9d, xe2x80x9cspring temperedxe2x80x9d or xe2x80x9ccold worked,xe2x80x9d are magnetic. Because stainless steel is corrosion resistant, it maybe used as a magnetic stirrer without any protective coating, like teflon, thus making stainless steel magnetic stirrers very inexpensive.
Magnetic levitation stirring is produced by placing the vessel with the stirrers in the middle of a strong laterally focused magnetic field. Thus the stirrers are pulled to the wall of the vessel and raised to the height of the center of the magnetic field. By raising or lowering the vessel or the magnetic field one can move the stirrers up and down within the vessel. You can also raise and lower the stirrers by moving the vessels laterally through a focused magnetic field or moving the magnetic field laterally past the vessels. This lateral movement method relies on gravity to pull the stirrers down to the bottom of the vessel after the vessel passes through the magnetic field. In cases where the liquid is viscous, a magnet located along the path of lateral movement but below the vessel may be used to xe2x80x9cpullxe2x80x9d the stirrers to the bottom of the vessel. Magnetic levitation has the advantage of being able to stir and aerate tall columns of liquids with ease as it can be effected by simple vertical or lateral motion of either the vessel or the magnetic field.
It is also the object of the machines, device and method to provide a robust, economical and practical mixing method to resuspend particulates, to dissolve a solid in a liquid, to mix two or more liquid reagents, to emulsify two or more liquids, to break up aggregates, break up filamentous organisms, stimulate the growth of microorganisms, break open cells, or to mix and immobilize magnetic beads in vessels or microplate wells. The positive physical displacement of the liquid and particles by the levitation action of the stirrer produces a robust mixing or movement of the liquids and solids in the well. In the case where extracts are dried in the bottom of wells to be later suspended, the direct physical contact between the stirrer and the extract is often critical in the dissolving of the extract. Physical contact is also important in mixing oil and water to make an emulsion. This direct physical contact cannot be duplicated by orbital shaking, vortexing, rocker platform or vibration. Furthermore, the power of the levitation action is such that it will stir even viscous material like pure glycerol, aggregates, emulsions and heavy suspensions of particulates that can not be stirred by the electronic stirring point system of the Variomag.
The lateral motion version of the invention (xe2x80x9cCarousel Versionxe2x80x9d) uses one or more magnetic dipole tunnels through which the vessels to be stirred are moved laterally on a carousel wheel. One of the advantages of the Carousel Version is that many vessels may be stirred by just one dipole magnet. Another is the simplicity of the motion used to affect the stirring. A third advantage is that if you use multiple dipoles you can alternate the polarity (S-N and N-S) of the dipole magnetic field and the stir devices would not only levitate but would xe2x80x9ctumblexe2x80x9d and reverse their physical orientation as they align to the magnetic polarity of alternating (S-N and N-S) dipole magnetic fields.
The vertical motion version of the invention (xe2x80x9cElevator Versionxe2x80x9d) uses a single massive magnetic dipole shaft in which the vessels (the elevator car) are raised and lowered to raise and lower the stirrers inside the vessels. One of the advantages of the Elevator version is that it is more compact and is able to use the same magnetic field to also pull the stirrer down in viscous liquids. Because of it""s compact nature it will fit into standard incubators so cultures can be stirred and aerated.
In both versions the speed of stirring and the amplitude of the stirring motion are easily controlled. Also the vigor of stirring action is also controlled by the size and shape of the stirrer placed in the vessel and can be changed to accommodate liquids of different viscosities or particulates of differing sizes.
We have also developed simple dispensing systems that will efficiently place the various stir devices (balls, discs, bars or dowels) into all the wells of a microplate (from the 6 well microplate to the 1536 well microplate) in a single step. Thus the technical process of placing stirrers in microplates for Magnetic Levitation Stirring is easy.
Also the process of removing the stirrers is simple. Just place a powerful magnet over the top of the microplate or a replicator with magnetic pins to extract the stirrers. Furthermore unlike the single 96 well microplate that a Hydra work station pipetter can mix only one microplate at a time, the Carousel Version Stirrer is able to mix 12 or more microplates simultaneously as they pass through levitation tunnels and pull down stations. The only limitation to the number of vessels that can be stirred is the diameter of carousel that can be accommodated. Furthermore the Hydra work station pipetter must wash each of the pipets between microplates to avoid contamination. Although pipetting the contents of wells up and down will provide adequate mixing and aeration, this is a very laborious, cumbersome operation that can not be done in processes that require continuous mixing such as microbial culturing.
Carousel Magnetic Levitation Stirrers cost under $22,500 and the stir balls, discs, bars and dowels are very inexpensive (from $0.03 to $0.15 each). Furthermore the stirrers can be reclaimed, washed, sterilized and reused over and over again because they are made from stainless steel. Thus the setup cost of tumble stirring for twelve 96 well microplates including stirrers is $1,932.60 compared to $2,635.00 for the Variomag and $30,000 for the Hydra pipetting work station. Furthermore it is not clear that the Variomag will even stir deep well microplates as the bottom of these wells are cup shaped and have ridges and there are no reports or claims that the Variomag will stir deep well microplates.
It is also an object of this device to be small enough to fit inside standard culture incubators so that microbial cultures can be stirred while growing. The Elevator Version of the Magnetic Leviatation Stirrer is 12xe2x80x3 wide by 17xe2x80x3 long by 16xe2x80x3 high and thus will fit into the 18xe2x80x3 wide door on most culture incubators.
It is also an object of this device to be made as large or as small to fit the application at hand.
It is also an object of this device to have a speed controller attached to the device so as to determine the speed or vigor of mixing.
Still further objects and advantages will become apparent from a consideration of the ensuing description and accompanying drawings.